CN102791753B - Polymers comprising a majority of amphiphilic monomers intended for trapping and manipulating membrane proteins - Google Patents
Polymers comprising a majority of amphiphilic monomers intended for trapping and manipulating membrane proteins Download PDFInfo
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- CN102791753B CN102791753B CN201080057496.6A CN201080057496A CN102791753B CN 102791753 B CN102791753 B CN 102791753B CN 201080057496 A CN201080057496 A CN 201080057496A CN 102791753 B CN102791753 B CN 102791753B
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Abstract
The invention relates to an amphiphilic polymer comprising at least 75% of amphiphilic monomers of formula (I), the average molar mass of the polymer being between 800 and 100 000, and also water-soluble complexes between the polymer according to the invention and a hydrophobic or amphiphilic compound, in particular a membrane protein, a concentrated aqueous solution of one or more such complex(es), a product comprising one or more such complex(es) attached to a substrate by means of the polymer according to the invention, and various uses of these products.
Description
Technical field
The present invention relates to the amphipathic nature polyalcohol for manipulating hydrophobic compound in aqueous, the water-soluble compound formed between hydrophobic compound (particularly membranin) and described polymkeric substance, for the preparation of the method for described mixture, and the application of described mixture, be applied to diagnosis or analytical procedure especially.
Background technology
AQP-CHIP (a kind of protein of particular variety) is inserted in testing in vivo in microbial film (described AQP-CHIP is through the double resin layer of described film).The surface (trans-membrane region) of these protein naturally contacted with film is hydrophobic especially, and for them, the surface outside film is mainly hydrophilic.Membranin ensure that basic biological function, particularly about the exchange of information or molecule between various cellular compartment and between cell and environment thereof.
In this regard, membranin has the main interest be in medical field.Such as, they represent the target enjoyed privilege of drug molecule.They also relate to many human diseasess, some disease wherein (such as multiple answer sclerosis or myasthenia gravis (myasthenia gravis)) has autoimmunization composition, and described autoimmunization composition is shown by the autoantibody for membranin be present in blood.
Manipulate membranin in aqueous usually their purifying and the research of their structure and function to be absolutely necessary prerequisite.Its make to avoid hydrophobic domains spontaneous polymerization and, for this purpose, the amphipathic environment maintained near trans-membrane region becomes required.
The conventional formulation of protein such in water soluble state contains the super micella enriched material of special surface promoting agent, sanitising agent.The success of method adsorbs these amphiphilic cpdss and dispersion agent based on transmembrane protein region.But exactly due to the existence of sanitising agent, the manipulation of the mixture formed thus is much more exquisite than the manipulation of soluble proteins.Comprise in the solution of studied protein all, when concentration is greater than its micelle-forming concentration (cmc), said circumstances will inevitably occur.Except the possible Cost Problems caused by sanitising agent consumption, due to the fact that membranin is normally unstable in detergent solution, experiment will be implemented usually fine.Therefore, under the existence of excessive micella, they have the trend of irreversibly sex change, and the defect of tensio-active agent causes them to precipitate usually.
This situation has caused the surrogate found sanitising agent and use, wherein will quote such as duplicature micella, described duplicature micella is by little lipid disk---the nanometer disk of surfactants stabilize, its similar is in the series of the molecule institute subordinate of formation present patent application theme, but tensio-active agent is wherein protein---clean dose of peptide (peptitergents), clean dose of described peptide is peptide amphiphile, lipopeptid, carry hydrocarbon chain peptide, fluoridize or half fluorinated surfactant and amphipathy macromolecule.
Amphipathy macromolecule is amphipathic nature polyalcohol people such as (, WO 1998/027434) Tribet that the cross-film surface of being intended to membranin especially substitutes sanitising agent.This patent describes and is total to amphipathic nature polyalcohol for maintaining the purposes of the membranin in water-bearing media.
Most of amphipathy macromolecule described up to now or so-called molecule are Ionomers, particularly anionic, therefore and the crystallization of stable membranin is not favorable factor this forbids that they are in various analysis (isoelectrofocusing) or the application that is separated in (chromatography on ion exchange column) system, and this is for.Therefore need to have the amphipathic nature polyalcohol that is better than existing amphipathy macromolecule to manipulate membranin and it will be non-ionic type.
Non-ionic type amphipathy macromolecule has been described in the people such as people and Sharma such as Prata.In the people such as Prata, amphipathy macromolecule is the multipolymer (Fig. 2 see this file) comprising two type monomers, and a kind of is hydrophilic (2 OH and 1 sugar or 3 OH), and another kind is amphipathic (2 OH and 1 aliphatic chain).In this document, the mol ratio between hydrophilic monomer and amphiphilic monomer has been maintained between 3.0 and 6.7, and it is equivalent to the hydrophilic monomer of 75-87% and the amphiphilic monomer of 13-25%, and therefore described amphiphilic monomer is minority.
In the people such as Sharma, amphipathy macromolecule is the multipolymer (scheme 1 see this file) comprising two type monomers, and a kind of is hydrophilic (2 OH and 1 sugar), and another kind is amphipathic (1 OH, 1 sugar and 1 aliphatic chain).In this document, the mol ratio between hydrophilic monomer A and amphiphilic monomer B is maintained between 3 and 5, and it is equivalent to the amphiphilic monomer B of hydrophilic monomer A and 17-25% of 75-83%, and therefore described amphiphilic monomer is minority.This is interpreted as the following fact: author has been noted that the amphipathy macromolecule of the amphiphilic monomer comprising the highest per-cent (25%) has had the water-soluble of reduction.
Patent application WO 2008/058963 describes the fixing of the membranin on carrier of the method by amphipathy macromolecule, described amphipathy macromolecule is the multipolymer comprising dissimilar monomer (hydrophilic, amphipathic, hydrophobic), and wherein the percent of total of hydrophobicity or amphiphilic monomer is contained in (claim 3 see WO 2008/058963) between 0.25 and 2.5 relative to the ratio of hydrophilic monomer percent of total.Exemplary amphipathy macromolecule is the ionic copolymer (Figure 1A see this file) comprising hydrophilic monomer and hydrophobic monomer.In addition, be defined as hydrophilic and hydrophobic functional group that amphipathic group is included in mixing in identical " grafting (greff é) " in this application, and be not grafted on hydrophilic radical on side chain and hydrophobic grouping regardless of separating.
Therefore, all amphipathy macromolecules described up to now or so-called molecule are multipolymers, and it comprises unit of different nature, and some are hydrophilic, and other is hydrophobic and/or amphipathic, and when amphiphilic monomer exists, it is minority.
Further, the prompting of the result that proposes in the people such as Sharma, except amphiphilic monomer, is necessary to comprise hydrophilic monomer, to keep enough water-soluble of amphipathy macromolecule.
But the copolymeric structure of all molecules described up to now has serious shortcoming: this makes accurately to reappear identical chemical structure from one batch to next batch and becomes difficulty.In fact, synthesize or need free-radical polymerized effect, or needing the functionalized at random of homopolymer type precursor, it is two kinds of non-selective reaction types in essence.Therefore, need and be better than those amphipathic nature polyalcohol of prior art for manipulating hydrophobic compound, particularly membranin, and it is much higher from the circulation ratio of the preparation of batch to next batch.
Summary of the invention
From point out in the people (4) such as Sharma different, the present inventor illustrates the equal amphipathic nature polyalcohol (homoAPols) be made up of amphiphilic monomer in astonishing mode, or the multipolymer (" standard-homopolymer ") comprising the such amphiphilic monomer of major part can have enough water-soluble to make it possible to manipulation membranin and copolymerization amphipathy macromolecule well known in the prior art.And, these homopolymer or standard-homopolymer can be produced in complete reproducible mode, and therefore not there is the shortcoming of copolymerization amphipathy macromolecule well known in the prior art.
Therefore, the application relates to a kind of amphipathic nature polyalcohol, and it comprises at least 75%, at least 80%, advantageously at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% the amphiphilic monomer of formula (I):
Wherein
R
1and R
2independently selected from H or C
1-C
3alkyl group (preferable methyl);
X and Y is independently selected from Sauerstoffatom, sulphur atom, acyloxy (-(CO) O-) or oxygen carbonyl (-O (CO)-) group, urethane group (-OCONH-) and formula (-CONR
6-) or (-NR
6cO-) amide group, wherein R
6hydrogen atom or C
1-C
6alkyl (preferable methyl or ethyl);
R
3and R
4independently selected from:
A) glucosides group,
B) zwitter-ion residue,
C) formula-(O (CH
2)
x)
ypoly-(alkylidene oxide) group of-OH, wherein x is contained in (advantageously, x equals 2) between 1 and 6, and y is contained between 4 and 30, advantageously, between 4 and 20 or between 4 and 10,
D) formula-(CH
2)
ncONR
7r
8or-(CH
2)
nnR
7cOR
8alkylamide group, wherein n is contained between 1 and 4, R
7and R
8independently selected from hydrogen atom (-H), C
1-C
6alkyl group (preferable methyl), glucosides group, zwitter-ion residue or formula-(O (CH
2)
x)
ypoly-(alkylidene oxide) group of-OH, wherein x to be contained between 1 and 6 (advantageously, x equals 2) and y is contained between 4 and 30, advantageously, between 4 and 20 or between 4 and 10,
R
5ring-type (R
5one or two can be contained, saturated or unsaturated ring, particularly hexanaphthene, pentamethylene or fragrant type) or acyclic (straight chain or side chain), saturated or unsaturated (one or more degree of unsaturation) comprises the hydrocarbon chain of 5 to 16 carbon atoms, or formula C
tf
2t+1(CH
2)
mpartly fluoridize hydrocarbon chain, wherein t to be contained between 2 and 10 and m is contained between 2 and 10,
The average molar mass of polymkeric substance is contained between 800 and 100000, and this is equivalent to the number of monomers be contained between 1 and 120, and advantageously, described average molar mass is below 50000 or equal 50000, preferably between 8000 and 50000.Average molar mass is calculated by weight.
" C
x-C
yalkyl " refer to formula-C
jh
2j+1, straight chain or side chain, saturated alkyl, wherein x<j<y.Especially, C
1-C
6alkyl can be C
1(methyl), C
2(ethyl), C
3(n-propyl or sec.-propyl), C
4(normal-butyl, isobutyl-, sec-butyl or the tertiary butyl), C
5(such as: n-pentyl, neo-pentyl, isopentyl, tert-pentyl) or C
6(such as n-hexyl) alkyl.
When polymkeric substance according to the present invention comprises other monomer of those monomers of the formula of being different from (I), described monomer is the monomer with vinylformic acid or ethene functional group, and described functional group has the side chain replaced by hydrophilic or hydrophobic grouping.Especially, the hydrophilic or hydrophobic grouping of described side chain can be selected from those groups of definition in the claim 3 and 4 of PCT International Publication WO 2008/058963, the content of these claims is incorporated to herein with the form quoted.
Advantageously, R
1and R
2independently selected from H or methyl group.More advantageously, R
1and/or R
2it is hydrogen atom.
Equally advantageously, X is Sauerstoffatom.
Equally advantageously, Y is urethane group (-OCONH-).
" glucosides group " refers to any group comprising sugar.Especially, for R
3and/or R
4favourable glucosides group be:
-mono--or two-sugar, or
The list-of-amination or two-sugar.
" monose " or " simple sugars " refers to the carbohydrate monomer of non-hydrolytic.Advantageously, described monose is selected from hexose (having the simple sugars of 6 carbon atoms), especially, is selected from glucose, seminose, semi-lactosi, allose, altrose, idose or maltose.
" disaccharides " or " disaccharide " refers to the sugar that two simple sugars by being connected by chemistry route (using the gelled acid of heat) or the glycosidic bond be hydrolyzed by enzymatic pathway are formed.Advantageously, described disaccharides is selected from two hexoses formed by two hexoses, as lactose (semi-lactosi β (l → 4) glucose), cellobiose (glucose β (l → 4) glucose) or maltose (glucose α (l → 4) glucose).
" polysaccharide " refer to by straight chain or sugar that the polymkeric substance of side chain is formed, described polymkeric substance is formed by the monomer that at least 2 are selected from monose as defined above and can be reached 20 unit, as some β-amylose.Therefore, term polysaccharide comprises disaccharides (or disaccharide), trisaccharide (or ternary sugar) etc., until 20 monosaccharide units.Preferably, described monosaccharide unit is hexose as defined above.
" list of amination-, two-or many-sugar " refers to any monose as defined above, disaccharides or polysaccharide, and wherein one or more alcohol functional group (-OH) are by amine (-NH
2) replace.Glucosamine, GalN, fructosamine or mannosamine can be cited as the example of the monose of amination, and amino Saccharum lactis is as the example of disaccharides.
Particularly preferably single-or the list-of two-sugar, particularly glucose, seminose, semi-lactosi, lactose, allose, altrose, idose, lactose, maltose or cellobiose type or two-hexose; Particularly preferably glucose, seminose and semi-lactosi, especially glucose.
Especially, when X is Sauerstoffatom, these glucosides groups or the oxygen grafting passed through on end group carbon (O glycosylation), or by the oxygen grafting of primary hydroxyl group (ester bond) or functional group's (amido linkage) grafting by amination, or eventually through the grafting of nitride group, wherein said end group carbon provides in advance in the replacement of oh group.In the later case, to be incorporated into by sugar on the propargyl functionalities grafting on X functional group in advance by Huygens's reaction (r é action de Huygens), in these Special Circumstances, described X functional group will be Sauerstoffatom.Advantageously, described glucosides group is by the oxygen grafting of end group carbon (O glycosidation).
" zwitter-ion residue " refers to have and is usually located on non-conterminous atom, and symbol is contrary, the group of the normal electric charge of a unit.These compounds have positivity and negative charge simultaneously, and they are highly soluble in water, and described water is polar solvent.Such as, favourable zwitter-ion residue is derived from simple betaines (particularly-N
+(CH
3)
2c (CH
2)
icO
2 -,-N
+(CH
3)
2c (CH
2)
isO
3 -, N
+(CH
3)
2c (CH
2)
ioSO
3 -type, wherein i is contained between 1 and 10), or be derived from amino acid functional group, especially, as there is polymerisable acrylic acid groups as CH
2=CHCONH-(CH
2)
j-, wherein j is contained in Methionin, ornithine, aspartic acid or the L-glutamic acid between 2 and 5.
In an advantageous embodiment, R
3and/or R
4glucosides group, the list-of preferred monose or disaccharides or amination or two-sugar, advantageously single-or two-sugar.Preferably, described list-or two-sugar are list-or two-hexose, the particularly list-of glucose, seminose, semi-lactosi, lactose, allose, altrose, idose, lactose, maltose or cellobiose type or two-hexose, advantageously glucose, seminose or semi-lactosi, more preferably glucose.
Advantageously, R
5ring-type (R
5one or two ring can be contained, saturated or unsaturated, the particularly ring of hexanaphthene or pentamethylene type) or acyclic (straight chain or side chain), saturated or unsaturated (one or more degree of unsaturation) hydrocarbon chain, advantageously straight chain and/or saturated, comprise the hydrocarbon chain of 5 to 16 carbon atoms.Preferably, R
5c
5-C
16, preferred C
8-C
12, particularly C
11alkyl group, the advantageously alkyl group of straight chain.
More accurately, favourable polymkeric substance according to the present invention comprises at least 75%, preferably at least 80%, advantageously at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% the amphiphilic monomer of formula (II):
Wherein
R
1and R
2independently selected from H or C
1-C
6alkyl group, (preferable methyl), R
3and R
4it is glucosides group as defined above.
R
5ring-type (R
5one or two can be contained, saturated or unsaturated ring, particularly hexanaphthene or pentamethylene or fragrant type) or acyclic (straight chain or side chain), saturated or unsaturated (one or more degree of unsaturation) hydrocarbon chain, it comprises 5 to 16 carbon atoms as defined above.
Advantageously, R
3and R
4(identical or different, preferably identical) be list-or two-sugar, preferred list-or two-hexose, the particularly list-of glucose, seminose, semi-lactosi, lactose, allose, altrose, idose, lactose, maltose or cellobiose type or two-hexose, preferred glucose, seminose or semi-lactosi, advantageously, R
3and R
4glucose.
Advantageously, R
5c
5-C
16alkyl, preferred C
8-C
12, particularly C
11alkyl, the alkyl of preferred straight chain.
In an advantageous embodiment:
-R
3and R
4be identical and represent glucose, seminose or semi-lactosi, preferred glucose, and
-R
5c
5-C
16alkyl, preferred C
8-C
12, particularly C
11alkyl, the advantageously alkyl of straight chain.
A particularly advantageous polymkeric substance according to the present invention comprises at least 75%, preferred at least 80%, advantageously at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% the amphiphilic monomer of formula (III):
The monomer of formula as above (I), (II) or (III) is the monomer with vinylformic acid or ethylene unit, and it comprises a hydrophobic aliphatic chain and two hydrophilic radicals (glucosides group or zwitter-ion residue).The chemical reaction that they can be known by expert, as glycosylation, amidate action, or making for synthesis by isocyanic ester.The synthesis of the monomer of formula (III) is described in detail in embodiment.The synthetic route with Complete comparability can be used to come semi-lactosi or the seminose of grafting replacement glucose, and/or the aliphatic chain, particularly other alkyl chain any of the another kind of type of grafting.
Polymkeric substance according to the present invention mainly comprises amphiphilic monomer.In an advantageous embodiment, polymkeric substance according to the present invention is a kind of homopolymer, described homopolymer comprises the formula as above (I) of the homogeneous chain of formation of 100%, the monomer of (II) or (III), optionally at another group of end bonding of described chain.
Really, can be prepared by the polyreaction that (as THF, acetonitrile or methyl alcohol, preferred THF) is caused by radical initiator (as AIBN or benzoyl peroxide) in the anhydrous solvent being heated at least 60 DEG C according to polymkeric substance of the present invention.Advantageously, in its building-up process, the size of polymkeric substance controls by adding mercaptan type chain transfer agent, and the concentration of the latter controls the size of polymkeric substance for monomer ratio.Second meaning that this transfer agent exists is that its permission introduces specific group at the end of chain, and described specific group can be used due to its special property.Therefore, in this case, polymkeric substance according to the present invention is included in the specific group of polymkeric substance first-in-chain(FIC).Therefore, when relating to according to homopolymer of the present invention, it comprises the possibility that polymkeric substance first-in-chain(FIC) exists specifically different groups, and described specifically different radical source from chain-transfer agent, and can be modified subsequently.
Especially, group can be comprised at first-in-chain(FIC) (in other words, at one of its end) further, the contained R of described group according to polymkeric substance of the present invention
9the thiol functionalities of-S-, wherein R
9advantageously be selected from:
-(CH
2)
mcOOH, wherein m=1 to 11,
-(CH
2)
m-NH
2, wherein m=2 to 11,
-(CH
2)
m-X-R
10, wherein m=1 to 11; X=O, NH, COO, CONH, S, phosphoric acid ester P (O) (O-R
10)
2; And R
10be selected from H, CH
3, benzoyl or benzyl group, fluorescent agent (derivative etc. as NBD, fluorescein or rhodamine), vitamin H, comprise hexose straight chain or the polysaccharide (particularly trisaccharide) of side chain, radical scavenger is as nitrone or oxynitride type ring-type paramagnetism class.
-(CH
2)
m-CONH (CH
2)
ps-R
11, wherein m is contained between 1 and 10, and p is contained between 2 and 11, and R
11be selected from H ,-C (C
6h
5)
3, fluorescent agent is if NBD or fluorescein, radical scavenger are if nitrone or oxynitride type ring-type paramagnetism class, vinylformic acid or vinyl monomer are as the oligomer derivative of methyl acrylate, acrylamide, THAM, vinyl-acetic ester.
-(CH
2)
m-CO (OCH
2cH
2)
xoCO (CH
2)
ps-R
11, wherein m is contained between 1 and 10, and x is contained between 3 and 100, and p is contained between 2 and 11, and R11 is as defined above,
-(CH
2)
2-(-OCH
2cH
2)
q-O-R
10, wherein q=1 to 100, and R
10it is as defined above,
-(CH
2)
rcONHC (CH
2oR
12)
3,-CH
2cONHC (CH
3) (CH
2oR
12)
2or CH
2cONHCH (CH
2oR
12)
2, wherein r is contained between 1 and 11, and R
12be selected from H, benzyl group or benzoyl group, fluorescent agent (derivative as NBD, fluorescein or rhodamine), the monose of vitamin H, optionally amination or straight chain or the polysaccharide (being preferably made up of the monomer of seminose, semi-lactosi, glucose, sialic acid, glucosamine, GalN and/or mannosamine) of side chain, radical scavenger is as nitrone or oxynitride type ring-type paramagnetism class.
-(CH
2)
mp (O) (OR
13)
2, wherein m is contained between 2 and 11, and R
13represent the C optionally replaced
1to C
16the alkyl group of straight chain,
-comprise 3 to 20 carbon atoms, saturated or unsaturated, optionally replace, the hydrocarbon chain of the straight chain particularly replaced by one or more OH group, the advantageously optional C replaced by one or more OH group (such as phytol)
3-C
20the alkyl group of straight chain or C
3-C
20the thiazolinyl of straight chain, or
-Shi C
tf
2t+1(CH
2)
mperfluorination chain, wherein t is contained between 2 and 10, and m is contained between 2 and 10.
All these sulfur alcohol compounds or can be commercially available, or the chemical reaction preparation that can easily pass through simple, high yield.
" C
3-C
20the thiazolinyl of straight chain " refer to and comprise 3 to 20 carbon atoms and the hydrocarbon chain comprising the straight chain of at least one double bond.
Advantageously, R
9expression-(CH
2)
rcONHC (CH
2oR
12)
3,-CH
2cONHC (CH
3) (CH
2oR
12)
2or-CH
2cONHCH (CH
2oR
12)
2, wherein r is contained between 1 and 11, wherein R
9represent H, benzyl or benzoyl group, fluorescent agent (derivative as NBD, fluorescein or rhodamine), the monose of vitamin H, optionally amination or straight chain or the polysaccharide of side chain, the compound of the monomer of preferred seminose, semi-lactosi, glucose, sialic acid, glucosamine, GalN and/or mannosamine.
Particularly preferred chain-transfer agent is R
9for (CH
2)
2cONHC (CH
2oH)
3chain-transfer agent.
When polymkeric substance according to the present invention comprises the monomer of formula (I) of 100%, this causes the polymkeric substance of formula (IV) subsequently:
Wherein R
1to R
5and R
9as defined above, and n is so to such an extent as to polymkeric substance has the average molar mass be contained between 800 and 100000, it is equivalent to n and is contained between 1 and 120, advantageously, described average molar mass below 50000 or equal 50000(n below 60 or equal 60), preferably between 8000 and 50000 (n is contained between 1 and 60).
A polymkeric substance very particularly preferably according to the present invention is formula V:
Wherein n is contained between 1 and 120, preferably between 1 and 60.
The invention still further relates to preparation according to the method for amphipathic nature polyalcohol of the present invention, it comprises foregoing formula (I), the monomer of (II) or (III) and chain-transfer agent in the presence of radical initiators, at least 60 DEG C, and the reaction in anhydrous solvent.
Described chain-transfer agent is sulfur alcohol compound, the compound of preferred formula (VI):
R
9-SH(VI), wherein R
9as defined above.
Especially, described radical initiator can be Diisopropyl azodicarboxylate (AIBN) or benzoyl peroxide.
The invention still further relates to hydrophobic or amphiphilic cpds, advantageously membranin, and according to the water-soluble compound of amphipathic nature polyalcohol of the present invention.Advantageously, described membranin is selected from film enzyme, membrane receptor, film ionic channel, the membrane antigen of microorganism or tumour, and medical protein (especially, as antibody).In addition, can be freezing or lyophilized form according to mixture of the present invention.
The invention still further relates to and there is one or more aqueous solution according to mixture of the present invention, the concentration of wherein said mixture is at more than 1g/l, advantageously at 2g/l, 3g/l or more than 4g/l, preferably at 5g/l, 6g/l, 7g/l, 8g/l, 9g/l or more than 10g/l.Advantageously, described concentration is at below 500g/l.Preferably, the concentration of described solution is between 10 and 500g/l.
The invention still further relates to a kind of carrier and at least one of comprising according to the product of mixture of the present invention, described mixture is by fixing on the carrier according to amphipathic nature polyalcohol of the present invention.
Finally, the present invention relates to the purposes according to mixture of the present invention, the aqueous solution or product, it for detecting existence or the disappearance of the part of described hydrophobic or amphiphilic cpds in biological specimen.
Accompanying drawing explanation
Fig. 1 estimates size and the dispersity of amphipathic telomer (t é lomere) particle by filtration over a molecular sieve.The storage solutions (batch SS174) of the equal telomer of 100 μ L (homot é lomere) is diluted in 900 μ L Tris/HCL damping fluids (20mM Tris, 100mM NaCl, pH=8.5), and is injected in Superose 1210-300GL post.Carry out wash-out with Tris damping fluid and detect at 220nm place.Vo and Vr be the excluded volume of display column and cumulative volume (be respectively 7.53 and 19.9mL) respectively.Apparent stokes radius (rayon de Stokes apparent) is 2.6nm.In order to compare, analyze A8-35 type conventional anion amphipathy macromolecule sample (batch FGH20) under the same conditions.The apparent stokes radius of A8-35 particle is 3.15nm.
Fig. 2 estimates size and the dispersity of the amphipathic telomer mixture of tOmpA/ by filtration over a molecular sieve.With two different protein/polymer quality ratio 1:4(central peaks) or 1:10(on the right side of peak) caught the membrane spaning domain (tOmpA) of the protein OmpA of the adventitia of bacteria Escherichia coli by amphipathic equal telomer, by described diluted sample (20mM Tris in Tris/HCl damping fluid, 100mM NaCl, and be injected in Superose 1210-300GL post pH=8.5).Carry out wash-out with Tris damping fluid and detect at 280nm place.Peak has been corrected as identical maximum value.The excluded volume of Vo and Vr difference display column and cumulative volume.In order to compare, the analysis A8-35 type conventional anion amphipathy macromolecule tOmpA sample (peak, left side) of catching under the same conditions.Elution volume is 11.9,12.2 and 12.6mL from left to right; The peak width at half height at peak is respectively 1.00,1.13 and 0.89mL.
Fig. 3 A8-35 or the equal telomer of nonionic catch after the UV/ visible absorption spectrum of bacteriorhodopsin.BR is caught, described amphipathy macromolecule or A8-35(batch of 5FGH20 with protein/amphipathy macromolecule mass ratio 1:5; Solid black lines), or the equal telomer of nonionic (batch SS174: grey filled lines; Batch SS298: black dotted lines).(with BioBeads in 4 DEG C of cultivation 2h, in the centrifugal 30min of 16.000 × g) this spectrum of immediate record after elimination sanitising agent.
Embodiment
The preparation of the equal amphipathic nature polyalcohol of embodiment 1.
1.1
the synthesis of disaccharides glycosidation acrylamide monomer: N-(1,1-bis-(O-β-D-Glucopyranose yloxymethyl)-1-(undecyl carbamyl yloxymethyl) methyl) embodiment of acrylamide.
According to first method, described method has produced the monomer being used to remaining embodiment, according to following scheme 1, from the THAM(be purchased its can by three-(methylol) aminomethanes with more than 90% yield obtain), synthesis experience three steps.
Scheme 1. synthesizes the first method of disaccharides glycosidation acrylamide monomer
Reagent and reaction conditions: a) (CH
3)
2c (OCH
3)
2, CH
3cN, tosic acid (apts), 20 °, R=80%; CH
3(CH
2)
10nCO, DABCO, toluene, 80 DEG C, R=98%; MK-10 resin, 48 hours, CH
2c1
2, 84%; HgCN
2, calcium sulfate, toluene, acetobromglucose (3 equivalent)))), r=63%
The synthesis of isopropylidene THAM
First, by envrionment temperature, in acetonitrile, under the tosic acid of catalytic amount exists, with Propanal dimethyl acetal process THAM 24 hours, close two hydroxy functional groups with the form of isopropylidene group.After normal processing, isopropylidene THAM crystallization is also separated with the yield of 80%.
5-acrylamido-5-undecyl amino frame formyl yloxymethyl-2,2-dimethyl-ring 1,3 Dioxaoctane
By isopropylidene THAM(2.64g, 12.28mmol, 1.0 equivalents) and Isosorbide-5-Nitrae-diazabicyclo [2,2,2] octane DABCO(4.05g, 14.74mmol, 1.2 equivalents) to be dissolved in dry toluene and mixture to be heated to backflow 30 minutes under argon gas.The toluene solution of dodecyl isocyanate (2.91g, 14.74mmol, 1.2 equivalents) is dropwise added in the solution maintaining 80 DEG C.After stirring 12 hours, add 5 methyl alcohol and mixture be placed in ethyl acetate (150mL).Use 1N HC1(3x100mL) and saturated NaCl solution (2x100mL) wash organic phase, at Na
2sO
4upper drying reduced under vacuum are to obtain the isopropylidene THAM compound (5.0g, 12.12mmol, 98%) had by the undecyl chain of carbamate groups bonding of white powder.Rf ~ 0.7, ethyl acetate/hexanaphthene (7:3v/v).
1h NMR (CDC1
3δ 7.01 (s, 1H), 6.21 (dd, J=1.6 and 17.0Hz, 1H), 6.08 (dd, J=10.0 and 17.0Hz, 1H), 5.65 (dd, J=1.6 and 10.0Hz, 1H), 4.99 (m, 1H), 4.72 (d, J=12.1Hz, 2H), 3.62 (d, J=12.0Hz, 2H), 3.20 (q, J=6.7Hz, 2H), 1.62 (s, 3H), 1.48 (m, 2H), 1.42 (s, 3H), 1.27 (s, 18H), 0.89 (t, J=6.6Hz, 3H).
13C NMR(CDC1
3δ165.7,157.6(CO),131.4(CH),126.5(CH
2),98.5(C),64.9,60.5(CH
2),53.5(C),43.0,41.3,31.9,31.3,29.8,29.6,29.6,29.5,29.3,26.7(CH
2),26.6(CH
3),22.7(CH
2),21.0,14.1(CH
3)。
N-(1,1-(2'3 ', 4 ', 6 '-four-O-ethanoyl-β-D-glucopyranosyl oxygen methyl)-1-(undecyl carbamyl yloxymethyl)-methyl)-acrylamide.
Above-claimed cpd (5.0g, 12.12mmol) and MK-10 resin (30g) are stirred 48 hours in methylene dichloride (200mL), then resin is filtered on plug of celite and use methyl alcohol (2x100mL) to rinse.Organic phase is concentrated to obtain N-(1,1-dihydroxymethyl-1-(undecyl carbamyl yloxymethyl) methyl)-benzamide (3.8g, 10.2mmol, 84%) under vacuo.Under argon gas, this compound (2.0g, 5.37mmol, 1.0 equivalents), mercury cyanide (2.13g, 16.10mmol, 3.0 equivalents) and calcium sulfate are mixed in toluene.After ultrasonic 2 minutes, add bromination tetra-acetylated glucose TAGB(6.62g, 16.10mmol, 3 equivalents) and by ultrasonic for mixture 30 minutes.Then reaction mixture filtered over celite and use ethyl acetate (100mL) to rinse.In succession wash organic phase with saturated sodium hydrogen carbonate solution (2x100mL), water (100mL), the liquor kalii iodide (4x50mL) of 10%, saturated thiosulfate solution (4x50mL) and water (2x50mL).By organic phase at Na
2sO
4upper drying concentrating under reduced pressure, make gained crude product experience flash-chromatography, with ethyl acetate/hexanaphthene (3:7v/v) wash-out to obtain the expection monomer of white powder.（3.5g，3.39mmol，63%）。R
f~ 0.35, ethyl acetate/hexanaphthene (7:3v/v).Mp 58.0℃。[α
D 25]=-12.70(c,1,CH
2C1
2)。
1h NMR (CDC1
3) δ 6.92 (s, 1H), 6.24 (dd, J=1.4 and 16.0Hz, 1H), 6.04 (dd, J=10.0 and 16.9Hz, 1H), 5.64 (dd, J=1.4 and 10.0Hz, 1H), 5.3-4.9 (m, 7H), 4.5 (m, 2H), 4.4-3.9 (m, 10H), 3.71 (dt, J=2.4 and 7.3Hz, 2H), 3.16 (q, J=6.5Hz, 2H), 2.11,2.09,2.07,2.05,2.02 (5s, 24H), 1.34 (m, 18H), 0.89 (t, J=6.6Hz, 3H).
13C NMR(CDCl
3)δ170.8,170.7,170.7,170.2,169.6,169.5,169.5,169.5,165.7,157.2(CO),131.3(CH),126.6(CH
2),101.0,100.8,77.3,72.6,72.5,71.8,71.8,71.1,68.3,68.2,(CH),68.6,68.3,68.0,64.5,61.7,60.4(CH
2),59.6(C),41.2,31.9,29.8,29.6,29.3,26.9,26.8,26.8,22.7(CH
2),21.1,20.8,20.8,20.7,20.7,20.6,20.6,20.6,14.2(CH
3)。HRMS (ESI
+) calculated value C
47h
72n
2o
23([M+H]
+): 1033.4599 measured values: 1033.4609 [M+H]
+.
According to optional method; according to following scheme 1; from the THAM(be purchased its can by three-(methylol) aminomethanes with more than 90% yield obtain); two steps are experienced in the synthesis of acrylamide monomer (N-(1,1-bis-(-O-β-D-glucopyranosyl oxygen methyl)-1-(undecyl carbamyl yloxymethyl) methyl) acrylamide).
Scheme 2. synthesizes the second method of disaccharides glycosidation acrylamide monomer
Reagent and reaction conditions: a) THAM (5 equivalent), CH
3(CH
2)
10nCO (1 equivalent), DABCO (0.5 equivalent), DMF, 60 DEG C, 3 hours, R=80%; B) HgCN
2, calcium sulfate, toluene, acetobromglucose (3 equivalent)))), r=63%
N-1,1-bis-(hydroxymethyl, methyl)-1-(undecyl carbamyl yloxymethyl)-methyl) acrylamide
Under an argon, THAM(21.9g in the 40mL DMF being heated to 60 DEG C, 125mMol, 5 equivalents) and diazabicyclo [2,2,2] octane (DABCO) (1.5g, 13.4mMol, 0.5 equivalent) stirred solution in dropwise add the undecyl isocyanic ester (5g, 25mMol, 1 equivalent) be dissolved in advance in 10mL methylene dichloride.Reaction mixture is maintained 60 DEG C until undecyl isocyanic ester all disappears (~ 30 minutes).Then the solvent was evaporated under reduced pressure also absorbs throw out with 200mL methylene dichloride.By suspension mechanical stirring 15 minutes under envrionment temperature.Retained sediments thing filtered and is again absorbed in the suspension of 100mL methylene dichloride, and then filtering once.Repetitive operation twice.By remaining throw out, in anhydrous methanol, recrystallization is to obtain 16.5g THAM immediately, and it can participate in reaction again.Merge organic phase, with the 1N HCl solution of 2 × 50mL, the saturated sodium carbonate solution of 2 × 50mL and the water washing of 2 × 50mL, drying over sodium sulfate, and concentrating under reduced pressure.By crude product in the solution of AcOEt/ hexane 2/8 crystallization to obtain the N-1 of white powder, 1-bis-(hydroxymethyl, methyl)-1-(undecyl carbamyl yloxymethyl)-methyl) acrylamide (7.55g, R=80%).R
f~ 0.5 (ethyl acetate/hexanaphthene (8:2v/v)
1h NMR (DMSOd
6) δ 7.56 (s, 1H), 7.12 (t, J=5,1H), 6.36 (dd, J=10 and 17.5Hz, 251H), 6.04 (dd, J=2.2 and 17.5Hz, 1H), 5.56 (dd, J=2.2 and 10Hz, 1H), 4.87 (m, 2H), 4.17, (s, 2H), 3.63 (s, 2H), 3.61 (s, 2H), 2.95 (m, 2H), 1.37 (m, 2H), 1.24 (m, 16H), 0.86 (t, J=6.75,3H).
13C NMR(DMSOd
6)δ166.5,156.8,132.8,125.5(CO),62.6,61.5,60.4(CH
2),31.8,29.9,29.5,29.2,29.3,26.7,22.6,14.5(CH
3)。
N-(1,1-(2'3 ', 4 ', 6 '-four-O-ethanoyl-β-D-glucopyranosyl oxygen methyl)-1-(undecyl carbamyl yloxymethyl)-methyl)-acrylamide
Under argon gas, above-claimed cpd (2.0g, 5.37mmol, 1.0 equivalents), mercury cyanide (2.13g, 16.10mmol, 3.0 equivalents) and calcium sulfate are mixed in toluene.After ultrasonic 2 minutes, add bromination tetra-acetylated glucose TAGB(6.62g, 16.10mmol, 3 equivalents) and by ultrasonic for mixture 30 minutes.Then reaction mixture filtered over celite and use ethyl acetate (100mL) to wash.In succession wash organic phase with saturated sodium hydrogen carbonate solution (2x100mL), water (100mL), the liquor kalii iodide (4x50mL) of 10%, saturated thiosulfate solution (4x50mL) and water (2x50mL).By organic phase at Na
2sO
4upper drying concentrating under reduced pressure, make gained crude product experience flash-chromatography, with ethyl acetate/hexanaphthene (3:7v/v) wash-out to obtain the expection monomer of white powder.（3.5g，3.39mmol，63%）。R
f~ 0.35, ethyl acetate/hexanaphthene (7:3v/v).Mp 58.0℃。[α
D 25]=-12.70(c,1,CH
2Cl
2)。
1h NMR (CDC1
3) δ 6.92 (s, 1H), 6.24 (dd, J=1.4 and 16.0Hz, 1H), 6.04 (dd, J=10.0 and 16.9Hz, 1H), 5.64 (dd, J=1.4 and 10.0Hz, 1H), 5.3-4.9 (m, 7H), 4.5 (m, 2H), 4.4-3.9 (m, 10H), 3.71 (dt, J=2.4 and 7.3Hz, 2H), 3.16 (q, J=6.5Hz, 2H), 2.11,2.09,2.07,2.05,2.02 (5s, 24H), 1.34 (m, 18H), 0.89 (t, J=6.6Hz, 3H).
13C NMR(CDCl
3)δ170.8,170.7,170.7,170.2,169.6,169.5,169.5,169.5,165.7,157.2(CO),131.3(CH),126.6(CH
2),101.0,100.8,77.3,72.6,72.5,71.8,71.8,71.1,68.3,68.2,(CH),68.6,68.3,68.0,64.5,61.7,60.4(CH
2),59.6(C),41.2,31.9,29.8,29.6,29.3,26.9,26.8,26.8,22.7(CH
2),21.1,20.8,20.8,20.7,20.7,20.6,20.6,20.6,14.2(CH
3)。HRMS (ESI+) calculated value C
47h
72n
2o
23([M+H]
+): 1033.4599 measured values: 1033.4609 [M+H]
+.
1.2
the synthesis of NAPol.
The synthesis (scheme 3) of telomer depends on the transfer agent derivative use of the thiohydracrylic acid with many benzoylations Tris group.These different benzoyl group have strong UV and absorb and the end being positioned at polymer chain.Therefore, they make it possible to quality and the mean polymerisation degree of the UV absorption Accurate Measurement telomer by measuring final product.Here it should be noted, the selection of tri-benzoyl functional group can be considered to introduce (character according to used transfer agent) interested functional group (fluorescein, cholesterol, vitamin H, nitrone etc.) and the example making chain end functionalized possibility.After this functionalized intermediary (as N-Hydroxysuccinimide, p-nitrobenzoic acid ester, pentafluorobenzoic acid ester etc.) telomerizing that also can occur in by being incorporated into the active ester groups on telogen in advance.
The synthesis of NAPol describes in following scheme 3:
The graphic extension of the equal telomer synthesis of scheme 3.
Reagent and condition: (a) AIBN (0.5 equivalent), THF, Ar, 66 DEG C, 24 hours, ~ 51%; (b) MeONa, MeOH, pH 8-9, envrionment temperature, 12 hours, after dialysis ~ 65%.
By monomer THAM; N-(1; 1-(2'; 3'; 4'; 6'-tetra--O-ethanoyl-β-D-glucopyranosyl oxygen methyl)-1-(undecyl carbamyl yloxymethyl)-methyl)-acrylamide (1.0g, 0.968mmol, 40.0 equivalents) is dissolved in THF(15ml) in.By advertising with argon gas solution is degassed and being heated to backflow 30 minutes.Then the telogen TA(12.62mg be dissolved in THF is added with microsyringe, 0.024mmol, 1.0 equivalents) and AIBN(1.98mg, 0.012mmol, 0.5 equivalent), existingly before the synthesis of described telogen TA to describe people such as () Sharma.Reaction mixture is stirred under reflux until monomer all disappears (~ 24 hours).Then it is concentrated under vacuo, and by using MeOH/CH
2cl
2the spatial exclusion chromatography of mixture (1:1, v/v) wash-out (
lH-20) coarse adjustment polymers is separated, then dry under vacuo.Be separated the protection state telomer (0.524g, 52%) of white powder.R
f=0.0 ethyl acetate/hexanaphthene (6:4v/v).
1h NMR (250MHz, CDCl
3δ ppm) the 0.8 (-CH of alkyl chain
3), 1.3-1.7 (alkyl chain-(CH
2)
10), 2.1-2.4 (wide s ,-OCOCH
3), the 3.1 (-NH-near methylene radical), 4.8-5.3 (m, glucose unit 2H, 3H, 4H, 5H and 6H), 6.6 (-NH), the 7.4-8.1 (C of three t, TA
6h
5).
Pass through
1equal telomer (2.0g, 1.91mmol) under an argon atmosphere, is dissolved in anhydrous methanol (50mL) after measuring molar mass by H NMR and UV.Add the sodium methylate MeONa of catalytic amount and solution is spent the night in envrionment temperature stirring.Then solution was neutralized (until pH=8) in 15 minutes with IRC 50 acidic resins by stirring.After filtration resin and evaporating solvent, telomer is experienced with the dialysis retaining the film being some 6-8KDa.By the polymkeric substance of lyophilization separation and purification, it obtains (0.850mg, 65%) with the form of white powder.
1h NMR (250MHz, DMSO-d
6, δ ppm) and the 0.8 (-CH of alkyl chain
3), the 1.2-1.6 ((-CH of alkyl chain
2)
10), the 3.2 (-NH-near methylene radical), 4.8-5.2 (m, glucose unit 2H, 3H, 4H, 5H and 6H), 7.1 (-NH).
The technology of exploitation is general and also can be applied to the monomer being connected with different sugar (particularly semi-lactosi and seminose) and the monomer being connected with fluorohydrocarbon chain.In addition, no matter amphipathic, hydrophobic or hydrophilic it can extend to the cotelomer being associated with all kinds monomer () easily, as before us for hydrophilic determined with the mixture of hydrophobic monomer.
The circulation ratio of the equal telomer that embodiment 2. obtains
According to the relative populations of monomer and telogen TA, synthesize different equal telomers.Synthesis condition and the chemical structure of the equal telomer of synthesis are summarized in table 1.
ainitial monomer/TA molar ratio,
b,cestimated by UV analysis
The synthesis condition of table 1. different N APol and chemical structure
In addition, with same precursor and the same terms, SS298 and SS325 is synthesized by several different batches.The circulation ratio analysis of SS298 batch is shown in in following table 2.
ainitial monomer/TA molar ratio,
b, cestimated by UV analysis
Show the circulation ratio of 2.SS298 batch
Result display is according to the excellent circulation ratio of amphipathic equal telomer of the present invention batch.
The characteristic of the equal telomer of embodiment 3. and physico-chemical property
In embodiment 3 and 4, the equal telomer of novel amphiphilic according to the present invention and reference substance amphipathy macromolecule A8-35 make comparisons, described A8-35 be as shown in the formula copolymerization anionic amphipathy macromolecule:
The chemical formula of scheme 4. amphipathy macromolecule A8-35.
After the hydrolysis of ester functional group, in embodiment 1 preparation with good grounds amphipathic equal telomer of the present invention all there is the solubleness of more than 100g/L in water.Solution is colourless and after vigorous stirring, produces a small amount of foam.Micelle-forming concentration (CMC) and critical aggregation concentration (CAC) can not be detected by stalagmometry, this shows, with A8-35 as a reference, CAC is extremely low.Small-angle neutron scattering measures (SANS; Do not show) and molecular sieve filtration (SEC; Fig. 1) show that the telomer of the type combines to obtain the particle of the total mass with 50kDa rank in aqueous, its associating in two telomer molecules about the same and close to before for the value that A8-35 type amphipathy macromolecule (~ 40kDa) measures.Effective radius can be comparable to the effective radius (~ 2.6vs. ~ 3.15nm) of A8-35 particle, is like this (Fig. 1) equally for dispersity.Observed by quasi-elastic light scattering (QLS), the solutions display of these telomers is formed by the particle of the uniform sizes of 5-6nm diameter, has excellent consistence (table 3) with SEC data.The size of particle to concentration (table 3) or temperature (table 4) very inresponsive.
Table 3. is measured in differing temps by QLS, and non-ionic type equal telomer SS174 particle is 10,50 and the diameter of 100g/L concentration.
aundetermined
Table 4. is measured in differing temps by QLS, and equal telomer SS298, SS293 and SS292 particle of non-ionic type is at the diameter of 50g/L concentration.
Therefore, the characteristic of the physico-chemical property of the amphipathic equal telomer of non-ionic type according to the present invention shows these amphipathy macromolecules and has the character being similar to copolymerization anionic amphipathy macromolecule A8-35.
The compound of the equal telomer of embodiment 4. and membranin
According to definition, amphipathy macromolecule is the amphipathic nature polyalcohol being used for keeping membranin solubility and biochemical stability under the disappearance of sanitising agent.The equal telomer of non-ionic type according to the present invention meets the ability of these two functions at two kinds of protein, and the trans-membrane region (tOmpA) of intestinal bacteria adventitia OmpA albumen and bacteriorhodopsin (BR) are tested.These two kinds of protein represent two kinds of main structure types that transmembrane protein adopts, β-pleated sheet structure ((tOmpA) and α spiral (BR).In addition, BR is protein relatively unstable in detergent solution, can measure its Denaturation easily by the release of its cofactor (retinene), the release of described cofactor causes the minimizing (disappearance of whole protein) of absorption near 564nm and the appearance (due to free retinene) at peak, 380nm place.
The data summed up in table 5 show, for be reduced to below sanitising agent micelle-forming concentration in detergent concentration after, keep two kinds of protein in the solution, two batches of equal telomers of test are in fact same effective with reference substance anionic amphipathy macromolecule A8-35, the concentration of described sanitising agent reduces or passes through not dilute containing the damping fluid of sanitising agent (tOmpA), or the absorption by polystyrene bead (BR): the retention rate in solution changes between 75 and 94%, compared to the 89-98% after the compound by A8-35, for the higher density of mixture of the difference (~ 75%vs. ~ 90%) observed by tOmpA probably owing to being formed with non-ionic type amphipathy macromolecule, it causes precipitation a small amount of in the high speed centrifugation process of the maintenance test be used as in solution.Lower speed is used for BR, this explains why maintenance difference less important (and protein precipitation-line 2-is more imperfect under the disappearance of amphipathy macromolecule) in solution.
Table 5. non-ionic type telomer maintains the ability of membranin in the solution
The equal telomer of non-ionic type of type described in scheme 3 above adding in the detergent solution of tOmpA and BR with shown mass ratio, or batch SS174(4-5), or batch SS298(6-7).After cultivating 20 minutes, with the damping fluid dilution tOmpA solution not containing sanitising agent, to make the concentration of sanitising agent be reduced to below cmc, but add polystyrene bead (BioBeads) in BR solution, sanitising agent is adsorbed on described polystyrene bead.After cultivating 2 hours, by solution in 200.000xg(tOmpA) or 16.000xg(BR) centrifugal 30 minutes.By measuring in 280nm(tOmpA, BR) place or in 554nm(BR) absorption at place estimates the protein portion that exists in supernatant liquor.Contrast comprises the diluent of protein example in detergent solution, described detergent solution is the detergent solution (1) with its more than cmc, or have not containing the detergent solution of damping fluid (2) of sanitising agent, and carry out with anionic amphipathy macromolecule A8-35(3 under the condition identical with experiment 4-7) catch experiment.N.d.: undetermined.
Primary data (not shown) shows that non-ionic type BR/ telomer mixture has the size that (in SEC) is comparable to BR/A8-35 mixture, and therefore it has purposes with it in biological chemistry and biophysics small size compatible mutually.Also be like this for non-ionic type tOmpA/ telomer mixture, present as clear in Fig. 2.
By the BR that catches in A8-35 or with the often a collection of uv-vis spectra in two batches of equal telomers of non-ionic type of test, the innocuousness of the equal telomer of (Fig. 3) non-ionic type relative to BR is described.In three kinds of situations, 554 and the specific absorption at 280nm place and the remarkable disappearance at peak, 380nm place show that protein is in its natural form and not yet discharges its cofactor.(sample of catching with SS174 batch absorption owing to slight turbidity) slightly higher in 280nm place.
In order to sum up, the biochemical test carried out makes it possible to determine: non-ionic type according to the present invention amphipathic equal telomer a) capture membrane albumen maintained and do not exist in the solvent of sanitising agent effectively; B) with the little mixture of its formation, the size of described mixture and dispersity are comparable to the mixture adopting anionic amphipathy macromolecule to be formed as A8-35; And c) stabilize membranin compared with detergent solution.In other words, these polymkeric substance have all characteristics, described characteristic make amphipathy macromolecule (and all application of the latter can be provided for) have its nonionic characteristic give their additional advantage, there is the height circulation ratio of synthesis, and dexterity, this dexterity make or can on each telomer chain on them the grafting single functional group determined, or as above A8-35 to be carried out, in a random way functionalized they.
Reference
Prata,C.,Giusti,F.,Gohon,Y.,Pucci,B.,Popot,J.-L.&Tribet,C.(2001).Non-ionic amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane keep membrane proteins soluble and native in the absence of detergent.Biopolymers 56,77-84.
Sharma,K.S.,Durand,G.,Giusti,F.,Olivier,B.,Fabiano,A.-S.,Bazzacco,P.,Dahmane,T.,Ebel,C.,Popot,J.-L.&Pucci,B.(2008).Glucose-based amphiphilic telomers designed to keep membrane proteins soluble in aqueous solutions:synthesis and physical-chemical characterization.Langmuir 24,13581-13590.
Tribet,C.,Audebert,R.&Popot,J.-L.(1996).Amphipols:polymers that keep membrane proteins soluble in aqueous solutions.Proc.Natl.Acad.Sci.USA 93,15047-15050.
WO 1998/027434
WO 2008/058963
Claims (25)
1. an amphipathic nature polyalcohol, it comprises the amphiphilic monomer of the formula (I) of at least 90%:
Wherein
R
1and R
2independently selected from H or C
1-C
6alkyl group;
X and Y is independently selected from Sauerstoffatom, sulphur atom, acyloxy (-(CO) O-) or oxygen carbonyl (-O (CO)-) group, urethane group (-OCONH-) and formula (-CONR
6-) or (-NR
6cO-) amide group, wherein R
6hydrogen atom or C
1-C
6alkyl;
R
3and R
4glucosides group, and independently selected from:
-mono--or two-carbohydrate, and
The list-of-amination or two-sugar; R
5c
5-C
16alkyl group;
With polymer weight, its average molar mass is contained between 800 and 100 000.
2. amphipathic nature polyalcohol according to claim 1, is characterized in that described R
1and/or R
2it is hydrogen atom.
3. amphipathic nature polyalcohol according to claim 1, is characterized in that described X is Sauerstoffatom or sulphur atom.
4. amphipathic nature polyalcohol according to claim 3, is characterized in that described X is Sauerstoffatom.
5. amphipathic nature polyalcohol according to claim 1, is characterized in that described Y is urethane group (-OCONH-).
6. amphipathic nature polyalcohol according to claim 1, is characterized in that described R
3and/or R
4be glucosides group, described glucosides group is selected from list-or the two-hexose of glucose, seminose, semi-lactosi, lactose, allose, altrose, idose, lactose, maltose and cellobiose type.
7. amphipathic nature polyalcohol according to claim 1, is characterized in that described R
3and/or R
4be glucosides group, described glucosides group is selected from glucosamine, amination semi-lactosi, fructosamine, amination seminose and amino Saccharum lactis.
8. amphipathic nature polyalcohol according to claim 6, is characterized in that described R
3and R
4glucose.
9. amphipathic nature polyalcohol according to claim 1, is characterized in that described R
5c
11linear alkyl groups.
10. amphipathic nature polyalcohol according to claim 1, is characterized in that:
R
1and R
2hydrogen atom,
X is Sauerstoffatom,
Y is urethane group (-OCONH-),
R
3and R
4be selected from single-or the list-of two-carbohydrate and amination or glucosides group of two-carbohydrate, and
R
5c
5-C
16alkyl group.
11. amphipathic nature polyalcohols according to claim 1, is characterized in that it comprises the monomer of the formula (III) of at least 90%:
12. amphipathic nature polyalcohols according to claim 1, is characterized in that it comprises the amphiphilic monomer of the formula (I) of 100%.
13. amphipathic nature polyalcohols according to claim 11, is characterized in that it comprises the amphiphilic monomer of the formula (III) of 100%.
14. amphipathic nature polyalcohols according to claim 1, is characterized in that it comprises a group at the end of chain further, the contained R of described group
9the thiol functionalities of-S-, R
9be selected from:
-(CH
2)
acOOH, wherein a=1 to 11,
-(CH
2)
b-NH
2, wherein b=2 to 11,
-(CH
2)
c-ZR
10, wherein c=l to 11; Z is selected from O, NH, COO, CONH, S and phosphoric acid ester P (O) (O-R
10)
2; And R
10be selected from H, CH
3, benzoyl group, benzyl group, fluorescent agent, vitamin H, comprise hexose straight chain or the polysaccharide of side chain and radical scavenger,
-(CH
2)
d-CONH (CH
2)
es-R
11, wherein d is contained between 1 and 10, and e is contained between 2 and 11, and R
11be selected from H ,-C (C
6h
5)
3, fluorescent agent, radical scavenger and vinylformic acid or vinyl monomer oligomerization derivative,
-(CH
2)
f-CO (OCH
2cH
2)
goCO (CH
2)
hs-R
12, wherein f is contained between 1 and 10, and g is contained between 3 and 100, and h is contained between 2 and 11, and R
12be selected from H and-C (C
6h
5)
3,
-(CH
2)
2-(-OCH
2cH
2)
q-O-R
13, wherein q is included between 1 to 100, and R
13be selected from H, CH
3, benzoyl group, benzyl group, fluorescent agent, vitamin H, monose and straight chain or the polysaccharide of side chain,
-(CH
2)
rcONHC (CH
2oR
14)
3,-CH
2cONHC (CH
3) (CH
2oR
14)
2or CH
2cONHCH (CH
2oR
14)
2, wherein r is contained between 1 and 11, and R
14be selected from H, benzyl group, benzoyl group, fluorescent agent, vitamin H, monose and straight chain or side chain polysaccharide,
-(CH
2)
ip (O) (OR
15)
2, wherein i is contained between 2 and 11, and R
15represent C
1to C
16the alkyl group of straight chain,
-straight chain, saturated or unsaturated, comprise the hydrocarbon chain of 3 to 20 carbon atoms, and
-Shi C
jf
2j+1(CH
2)
kperfluorination chain, wherein j is contained between 2 and 10, and k is contained between 2 and 10.
Amphipathic nature polyalcohol shown in 15. 1 kinds of formulas (V):
Wherein n is contained between 1 and 120.
16. amphipathic nature polyalcohols according to claim 15, wherein n is contained between 1 and 60.
17. 1 kinds of methods preparing the amphipathic nature polyalcohol according to any one of claim 1-16, the monomer of its contained (I) or (III) and chain-transfer agent are in the presence of radical initiators, in at least 60 DEG C, the reaction in anhydrous solvent.
The water-soluble compound of 18. 1 kinds of hydrophobic or amphiphilic cpds and a kind of amphipathic nature polyalcohols according to any one of claim 1-16.
19. water-soluble compounds according to claim 18, wherein said hydrophobic or amphiphilic cpds is membranin.
20. water-soluble compounds according to claim 19, is characterized in that described membranin is selected from the film enzyme of microorganism or tumour, membrane receptor, film ionic channel, membrane antigen and antibody.
21. water-soluble compounds according to claim 18, it is freezing or lyophilized form.
22. 1 kinds of aqueous solution with one or more water-soluble compounds according to any one of claim 18-21, the concentration of wherein said mixture is at more than 1g/l.
23. aqueous solution according to claim 22, it has the mixture of one or more between 10 and 500g/l of concentration according to any one of claim 18-21.
24. 1 kinds of products for the existence or disappearance that detect the part of hydrophobic or amphiphilic cpds in biological specimen, it comprises carrier and the mixture of at least one according to any one of claim 18-21, and described mixture is fixing on the carrier by the amphipathic nature polyalcohol according to any one of claim 1-16.
The purposes of 25. mixtures according to any one of claim 18-21, the aqueous solution according to claim 22 or 23 or product according to claim 24, it for detecting existence or the disappearance of the part of described hydrophobic or amphiphilic cpds in biological specimen.
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| FR0958072A FR2952642B1 (en) | 2009-11-16 | 2009-11-16 | POLYMERS COMPRISING A MAJORITY OF AMPHIPHILIC MONOMERS FOR TRAPPING AND HANDLING MEMBRANE PROTEINS |
| FR0958072 | 2009-11-16 | ||
| PCT/EP2010/067609 WO2011058195A1 (en) | 2009-11-16 | 2010-11-16 | Polymers comprising a majority of amphiphilic monomers intended for trapping and manipulating membrane proteins |
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| US (1) | US8642309B2 (en) |
| EP (1) | EP2501734B1 (en) |
| JP (1) | JP5306543B2 (en) |
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| EP2672269A1 (en) * | 2012-06-07 | 2013-12-11 | Solarwell | Enhanced affinity ligands |
| US9479298B2 (en) | 2013-07-08 | 2016-10-25 | Intel IP Corporation | Demodulation reference signals (DMRS)for side information for interference cancellation |
| KR101719122B1 (en) * | 2015-09-09 | 2017-03-23 | 한양대학교 에리카산학협력단 | Novel mannitol-based amphiphiles and uses thereof |
| TWI732825B (en) | 2016-05-20 | 2021-07-11 | 日商王子控股股份有限公司 | Oriented self-assembly composition for pattern formation, monomer for orientation self-assembly composition for pattern formation, and pattern formation method |
| WO2020115083A2 (en) * | 2018-12-03 | 2020-06-11 | Centre National De La Recherche Scientifique | Novel amphipols |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0896123A1 (en) * | 1997-08-05 | 1999-02-10 | Institut Français du Pétrole | Process to slow down the growth and/or the agglomeration and possibly delaying the formation of hydrates in a production effluent |
| CN1852930A (en) * | 2003-09-03 | 2006-10-25 | 罗地亚公司 | New copolymer having a controlled structure, and use thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54120696A (en) * | 1978-03-10 | 1979-09-19 | Takeda Chem Ind Ltd | Glucosamine derivative and its preparation |
| SE463314B (en) * | 1989-03-01 | 1990-11-05 | Biocarb Ab | COPOLYMERS OF AN N-ACYLED GLYCOSYLAMINE AND AN AMID, N-ACRYLOYL OR METACRYLYLYLYCOSYLAMINES, AND PROCEDURES FOR PREPARING THESE |
| DE69633742T2 (en) | 1996-12-16 | 2005-11-03 | Centre National De La Recherche Scientifique | WATER-SOLUBLE COMPLEX OF MEMBRANE PROTEINAMPHIPHILE ACRYLIC POLYMER AND USE FOR DIAGNOSIS |
| JP4552174B2 (en) * | 2002-12-13 | 2010-09-29 | 株式会社興人 | Urea derivative, process for producing the same, and polymer thereof |
| FR2873123B1 (en) * | 2004-07-13 | 2006-09-15 | Rhodia Chimie Sa | NOVEL GLYCOPOLYMERS, THEIR USES, AND MONOMERS USEFUL FOR THEIR PREPARATION |
| FR2908414B1 (en) | 2006-11-13 | 2012-01-20 | Centre Nat Rech Scient | IMMOBILIZATION OF MEMBRANE PROTEINS ON A SUPPORT THROUGH AN AMPHIPHILE MOLECULE |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0896123A1 (en) * | 1997-08-05 | 1999-02-10 | Institut Français du Pétrole | Process to slow down the growth and/or the agglomeration and possibly delaying the formation of hydrates in a production effluent |
| CN1852930A (en) * | 2003-09-03 | 2006-10-25 | 罗地亚公司 | New copolymer having a controlled structure, and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| 《Glucose-Based Amphiphilic Telomers Designed to Keep Membrane Proteins Soluble in Aqueous Solutions: Synthesis and Physicochemical Characterization》;K. Shivaji Sharma等;《Langmuir》;20081104(第24期);K. Shivaji Sharma等 * |
| 《Stabilization of polymerized vesicular systems: an application of the dynamic molecular shape concept》;Michel Wathier等;《Chemistry and Physics of Lipids》;20021231(第115期);17-37 * |
| 范潇.《STN检索报告》.《STN检索报告》.2014,1-32. * |
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| CA2780960C (en) | 2019-01-15 |
| EP2501734A1 (en) | 2012-09-26 |
| ES2553866T3 (en) | 2015-12-14 |
| EP2501734B1 (en) | 2015-09-02 |
| FR2952642A1 (en) | 2011-05-20 |
| CA2780960A1 (en) | 2011-05-19 |
| US20130005016A1 (en) | 2013-01-03 |
| CN102791753A (en) | 2012-11-21 |
| JP5306543B2 (en) | 2013-10-02 |
| WO2011058195A1 (en) | 2011-05-19 |
| US8642309B2 (en) | 2014-02-04 |
| FR2952642B1 (en) | 2012-01-06 |
| JP2013510588A (en) | 2013-03-28 |
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